Biomedical subjects

D Mercanti

Publications and source records attributed to D Mercanti.

At least 19 recordsLinked to original sources

PubMed2000

The chemokine growth-related gene product beta protects rat cerebellar granule cells from apoptotic cell death through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors.

Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K(+). We have found that the growth-related gene product beta (GRObeta) partially prevents the K(+)-depletion-induced cell death, and that the neuroprotective action of GRObeta on granule cells is mediated through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GRObeta-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 microM) treatment. Moreover, GRObeta-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GRObeta is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors.

2-Amino-5-phosphonovalerate↗

PubMed2000

UV-ozone ashing of cells and tissues for spatially resolved trace element analysis.

UV/ozone ashing of thin tissue sections and cell cultures is a simple technique to enhance relative elemental concentrations, while maintaining their spatial location at the sub-micron level. This approach may enhance the capability of spatially resolved analysis techniques to detect the distribution of trace elements in biological matrices. We present results from light microscopy and x-ray spectromicroscopy studies of tissues and cells demonstrating that the micro-structure is very well conserved. We show the signal enhancement resulting from the removal of carbon, which allows otherwise undetectable gadolinium to be mapped in cancer tissue for a novel neutron capture therapy.

Carbon↗

PubMed2000

Antagonists of P2 receptor prevent NGF-dependent neuritogenesis in PC12 cells.

The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represents a well-established cellular model system for studying NGF signalling. Interesting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptors, expressed in naive PC12 cells and recognised as important biological mediators of neurotransmitters and growth factors. We show here that Basilen Blue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurite outgrowth with an IC(50) in the 5-10 microM range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinoceptor ligands, are also effective in this regard. NGF-dependent regeneration and stability of neurites, selected NGF-dependent extracellular and intracellular protein phosphorylations, binding of [(3)H] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellular duplication, 5'-nucleotidase activity, NGF-induced tyrosine autophosphorylation of TrkA receptors are not affected. NGF furthermore directly modulates the extracellular release of ATP and especially the levels of P2X(2) receptor protein in PC12 cells. In addition, extracellular ATP improves the neuritogenic effect of sub-optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as useful tools to dissect different NGF-evoked functions, suggesting a mechanistic role for P2 receptors in the signalling pathways of NGF.

5'-Nucleotidase↗

PubMed2000

SDF-1alpha-mediated modulation of synaptic transmission in rat cerebellum.

The functional expression of the seven-transmembrane domain G protein-coupled chemokine receptor CXCR-4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti-CXCR-4 Ab, and by evaluating the calcium responses to the physiological agonist stromal-derived cell factor-1alpha (SDF-1alpha) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR-4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF-1alpha, both in cell bodies and in neuronal processes. Whole-cell patch-clamped PNs in cerebellar slices responded to SDF-1alpha application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF-1alpha-induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR-4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

The growth-related gene product beta induces sphingomyelin hydrolysis and activation of c-Jun N-terminal kinase in rat cerebellar granule neurones.

The growth-related gene product beta (GRObeta) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GRObeta receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GRObeta receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GRObeta activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GRObeta-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

Animals↗

PubMed1999

Cell ashing for trace element analysis: A new approach based on ultraviolet/ozone.

We studied a new approach to cell ashing based on illuminating the specimens with a low-pressure mercury discharge lamp. We analyzed with synchrotron spectromicroscopy its effects on different physiological elements in neurobiological specimens. Our results demonstrate that carbon is removed, whereas phosphorus, calcium, potassium, and sulfur are retained and their relative concentrations are enhanced. Applied to trace elements, this technique will enhance their practical detectability.

Animals↗

PubMed1998

CXC chemokines interleukin-8 (IL-8) and growth-related gene product alpha (GROalpha) modulate Purkinje neuron activity in mouse cerebellum.

We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.

Animals↗

PubMed1998

High sensitivity quantitative analysis of cobalt uptake in rat cerebellar granule cells with and without excitatory amino acids.

We quantified the effect of the excitatory amino acids kainate and glutamate on the uptake of cobalt in primary rat cerebellar granule neurons, by using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). We quantitatively demonstrated that Co2+ uptake, although enhanced by glutamate and kainate also takes place in the absence of excitatory amino acids. We also found that cobalt uptake is not significantly altered by the presence of glutamate receptor competitive or noncompetitive antagonists, indicating that cobalt uptake in granule neurons does not require glutamate receptor stimulation. Our results suggest, therefore, that Co2+ may enter the cell by passive diffusion through the plasma membrane.

Animals↗

PubMed1998

Neurofibromatosis type 2: growth stimulation of mixed acoustic schwannoma by concurrent adjacent meningioma: possible role of growth factors. Case report.

The authors report the case of a young man suffering from neurofibromatosis type 2 (NF2) who harbored bilateral acoustic schwannomas and a parasellar meningioma. Neuroimaging studies performed during a 4-year follow-up period showed that the bilateral schwannomas had grown very little and at similar rates. However, after the meningioma had infiltrated the tentorium and approached the ipsilateral schwannoma at the incisura, both Schwann cell tumors started to grow rapidly, particularly the one adjacent to the meningioma, of which the percentage of annual growth rate increased by approximately a factor of 10(2). At the same time, magnetic resonance imaging showed that this tumor also changed its features. During surgery, the acoustic schwannoma was firmly adherent to both meningioma and tentorium. Histological examination revealed meningotheliomatous cells in the schwannoma adjacent to the meningioma. Antiphosphotyrosine immunoblotting of PC12 cells was compatible with the presence of an epidermal growth factor (EGF)-like molecule in the cerebrospinal fluid (CSF) of the patient. This factor was not detected in the CSF of five other NF2 patients, two of whom bore associated bilateral acoustic schwannomas and meningioma in remote locations. It is hypothesized that the meningotheliomatous cells infiltrating the schwannoma triggered an autocrine/paracrine growth-stimulatory mechanism that involved an EGF-like factor.

Adolescent↗

PubMed1997

The effect of ashing on cells: spectromicroscopy of physiological elements.

We analyzed the effects of cold oxygen plasma ashing of neurobiological specimens on different elements with synchrotron spectromicroscopy. Our results demonstrate that while carbon is almost completely removed, phosphorus, calcium, potassium, sulfur, and, to some extent, nitrogen are retained and their relative concentration is enhanced.

Animals↗

PubMed1996

A Xenopus laevis homologue of the La autoantigen binds the pyrimidine tract of the 5' UTR of ribosomal protein mRNAs in vitro: implication of a protein factor in complex formation.

In Xenopus and other vertebrates, ribosomal protein mRNAs share a common sequence in the 5' untranslated region (5' UTR), in particular a pyrimidine tract at the 5' end, which has been demonstrated to be involved in the translational regulation of this class of mRNAs. In previous studies, carried out in the Xenopus system, we demonstrated the specific binding of two proteins (57 kDa and 47 kDa) to the pyrimidine tract of the mRNAs for three different ribosomal proteins. Here, we show that the two binding proteins are in fact one; one being the cleavage product of the other. By immunoprecipitation and protein purification, this binding protein has been identified as the Xenopus homologue of the human La autoantigen, an RNA-binding protein previously reported to be implicated in RNA polymerase III transcription termination and in translation initiation of poliovirus and immunodeficiency virus type 1 RNAs. We show that the specific interaction of La with the 5' pyrimidine tract of ribosomal protein mRNA is mediated by a protease-sensitive factor, which, after assisting La-RNA binding, dissociates from the complex and becomes again available to promote further binding. We show that mutations in the 5' UTR pyrimidine tract, known to disrupt the translational control of ribosomal protein mRNA, severely impair La binding. Although a direct relationship between ribosomal protein mRNA translation and La binding is not yet available, the properties of the interaction suggest that La protein, possibly together with other components, might be involved in translational regulation.

Animals↗

PubMed1996

A glutamate-sensitizing activity in conditioned media derived from rat cerebellar granule cells.

When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 microM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 x 10(5) cells/cm2) or increasing the volume by an identical 4-fold factor reduced cell death from 90-95% to 20-30%. Addition of a conditioned medium derived from high-density to low-density cultures or to high-volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate-sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1-7, 3T3 and CHP 100. Glutamate-sensitizing activity was not mimicked by trilodo-L-thyronine, insulin-like growth factor-I (IGF-I), truncated IGF-I, GPE [a tripeptide (gly-pro-glu) derived from IGF-I], brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor-alpha. However, IGF-I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate-sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triiodo-L-thyronine and BDNF did not support the production of glutamate-sensitizing activity. Glutamate-sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N-omega-nitro-L-arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate-sensitizing activity.

Analysis of Variance↗

PubMed1996

Do insulin-like growth factor binding proteins (IGFBPs) modulate the IGF-I growth promoting and differentiating effects in human neuroblastoma cells?

The insulin-like growth factors (IGFs) are known to stimulate both the proliferation and differentiation of neuroblastoma cells, but the role of the IGF binding proteins (IGFBPs) has not yet been established. In this study, human neuroblastoma SH-SY5Y cells have been treated with IGF-I and its potent analogue des(1-3)IGF-I alone or following preincubation with a differentiating agent such as 12-o-tetradecanoylphorbol-13-acetate (TPA). Cell proliferation and differentiation were evaluated. Conditioned medium was tested for the presence of IGFBPs by ligand blotting. The SH-SY5Y cell proliferation was maximally stimulated by des(1-3)IGF-I. The TPA-induced differentiation of SH-SY5Y, evaluated by assessment of cell morphology and GAP-43 expression as a biochemical marker of differentiation was potentiated by nanomolar concentrations of des(1-3)IGF-I and, to a smaller extent, IGF-I Conditioned medium showed the presence of a major IGFBP band with an approximate molecular weight of 32.5 kD and a very faint band of approximately 24kD. The IGFBP immunoblotting results suggest that the predominant band might represent IGFBP-2. Our data represent a first demonstration of the presence of IGFBPs in conditioned medium of human neuroblastoma SH-SY5Y cells. The finding that the potent IGF-I analogue des(1-3)IGF-I with reduced affinity for IGFBPs induce major effects on cell growth and differentiation suggests that the IGFBPs may play an active role in the neuronal response to the proliferative and differentiative effects of IGF-I.

Animals↗

PubMed1995

Neurone decapping characterization by atomic force microscopy: a topological systematic analysis.

We tested a new approach to cell decapping on rat cerebellar neurones, and observed its effects on cell topography by atomic force microscopy (AFM). The results clearly demonstrate the effectiveness of our decapping approach, and also the ability of AFM to reveal fine details of the decapped cells. Specifically, varying the conditions and duration of the decapping process modifies the extent of the decapping. Such a method can be used to investigate the cytoplasm with surface sensitive techniques.

Animals↗

PubMed1995

Atomic Force microscopy of neuron networks.

We imaged uncoated neuron networks by an atomic force microscope in the repulsive regime of contact mode. Images of granule cells and their axons have been clearly revealed with details smaller than 20 nm. The good stability of the sample and the mechanical reproducibility of the microscope allowed the imaging of a neuron culture area of several square microns. By combining tens of images, we were able to reconstruct a highly defined neuronal network. Furthermore, the images were very reproducible over repeated scanning acquisition, demonstrating the mechanical and thermal stability of the instrument-sample system.

Animal Population Groups↗

PubMed1994

Aluminium in rat cerebellar primary cultures: glial cells and GABAergic neurones.

Experimental evidence of the preferential uptake of aluminium by GABAergic neurones and glial cells was provided by synchrotron spectromicroscopy studies. We observed rat cerebellar cultures enriched for GABAergic neurones or glial cells exposed to aluminium ions, detecting the presence and identifying the chemical status of aluminium on cell structures.

Absorptiometry, Photon↗

PubMed1994

Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.

Animals↗

PubMed1993

Sodium, calcium and late potassium currents are reduced in cerebellar granule cells cultured in the presence of a protein complex conferring resistance to excitatory amino acids.

Whole-cell, patch-clamp recordings were used to study voltage-gated currents generated by cerebellar granule cells that were cultured in medium containing either 10% fetal calf serum (hereafter termed S + granules) or neurite outgrowth and adhesion complex (NOAC, hereafter called NOAC granules). NOAC is a protein complex found in rabbit serum that renders granules resistant to the excitotoxic action of excitatory amino acids. During depolarizing commands both S+ and NOAC granules generated Na+ and Ca2+ inward currents and an early and a late K+ outward currents. However, Na+ and Ca2+ inward currents and late outward K+ currents recorded in NOAC granules were smaller than those seen in S+ granules. Furthermore, although of similar amplitude, early K+ currents displayed different kinetics in the two types of neurons. Thus, these data demonstrate that the electrophysiological properties of cerebellar granules, and probably of other neuronal populations, depend upon serum components and raise the possibility that an analogous modulation might be operative in vivo, and play a role in development, synaptic plasticity or neuropathological processes.

4-Aminopyridine↗